Article Figures & Data
Figures
- FIG 1
Mutant p53 inhibits MDM2 E3 ligase activity. (a) MDM2 was cotransfected with p53 mutants in H1299 cells for 48 h. Protein expression was detected by Western blotting. *, the G245S mutant contained N-terminal FLAG tag. (b) MDM2 was cotransfected with p53 mutants and His6-ubiquitin in H1299 cells for 48 h. MDM2 self-ubiquitination was analyzed by Ni2+-NTA pulldown and Western blotting. (c) Dose-dependent inhibition of MDM2 self-ubiquitination by mutant p53. (d) MDM2 self-ubiquitination was abrogated by point mutation (H457S) or deletion of the RING domain. (e) MDM2 ubiquitination of MDMX was inhibited by mutant p53. (f) Purified MDM2-p53 or MDM2-R175H mutant complex was incubated with charged E2. The release of ubiquitin from E2 was detected by Western blotting.
- FIG 2
Construction of a cleavable MDM2 protein. (a) MDM2GP structure. PreScission cleavage site and epitope tags were inserted after residues 171, 332, and 422 of MDM2. (b, c) MDM2GP and MDM2 were cotransfected with p53 (b) or MDMX (c) in H1299 cells. The degradation of p53 and MDMX by MDM2 or MDM2GP was analyzed by Western blotting. (d) MDM2GP was immobilized on beads using anti-FLAG antibody and cleaved with PreScission for 1 h. SQ-RING and RING fragment dissociation from the immobilized AD was detected by HA Western blotting. IP, immunoprecipitation; Sup, supernatant. (e) MDM2GP was immobilized using HA antibody and cleaved with PreScission. AD fragment dissociation from the immobilized RING domain was detected by FLAG Western blotting. (f) MDM2GP was immobilized using anti-4B2 antibody and cleaved with PreScission. AD and RING fragment dissociation from the immobilized p53BD was detected by FLAG and HA Western blotting. (g) Model of intramolecular interaction between MDM2 AD and RING domain. SQ designates the region with multiple ATM phosphorylation sites (residues 386 to 429).
- FIG 3
Increased binding of mutant p53 to MDM2 AD and RING domains. (a) Diagram of proteolytic fragment release (PFR) assay for detecting intermolecular interactions. MDM2GP-p53 complex from transfected H1299 cells was immobilized using anti-p53 antibody Pab421 conjugated to protein A beads. MDM2GP was cleaved by PreScission on the beads, and the release of MDM2 fragments was detected by Western blotting. (b) Comparison of MDM2 domain interactions with wild-type (Wt) and mutant p53. Wild-type p53 or R175H mutant was cotransfected with MDM2GP in H1299. MDM2GP-p53 complex was immobilized by Pab421 beads and cleaved by PreScission. MDM2 fragments that remained bound to the beads or dissociated into the supernatant were analyzed by Western blotting (WB). (c) Comparison of MDM2 domain interactions with wild-type and additional p53 mutants using the fragment release assay.
- FIG 4
Mutant p53 binding to MDM2 AD in coexpression and pulldown assay. (a) The minimal AD (mAD, residues 230 to 260) was expressed as a fusion to Myc-tagged GFP. Myc-GFP-mAD was coexpressed with p53 in H1299 cells. The interaction of mAD with p53 was analyzed by IP-Western blotting. (b) H1299 cells were transfected with the MDM2 RING domain fragment (residues 361 to 491) and p53. The interaction of RING and p53 was analyzed by IP-Western blotting. (c) H1299 cells were transfected with GFP-mAD8GS (containing hydrophobic-to-hydrophilic substitution of 8 residues) and p53 mutants. The binding between mAD and p53 mutants was analyzed by IP-Western blotting.
- FIG 5
MDM2 AD and RING domains bind to p53 core domain. (a, b) Beads loaded with GST-p53 and GST-R175H deletion mutants were incubated with H1299 lysate containing MDM2GP to form p53/MDM2GP complexes that were then cleaved with PreScission. The release of MDM2 fragments from the p53 complex was detected by Western blotting. Numbers above lanes show p53 residues present in each construct. (c) Diagram of MDM2 AD and RING binding to p53 core domain. (d) H1299 cells cotransfected with MDM2GP and p53 were treated with 20 μM 17-AAG for 5 h. MDM2GP-p53 complex was captured on Pab421 beads and cleaved by PreScission. The release of MDM2 fragments from p53 complex was detected by Western blotting. (e) SJSA cells were cultured in medium containing 17-AAG (50 μM) for 24 h. The turnover of MDM2 and p53 was analyzed by Western blotting after treatment with cycloheximide (CHX; 100 μg/ml).
- FIG 6
Mutant p53 interferes with MDM2 RING function. (a) MDM2 or MDM2ΔAD (with deletion of residues 210 to 290) was coexpressed with mutant p53 and His6-ubiquitin in H1299 cells. MDM2 self-ubiquitination was analyzed by Ni2+-NTA pulldown and MDM2 Western blotting. (b) MDM2 or MDM2-Praja (MDM2 RING replaced with Praja RING) was cotransfected with p53 and His6-ubiquitin in H1299 cells. MDM2 self-ubiquitination was analyzed by Ni2+-NTA pulldown and MDM2 Western blotting.
- FIG 7
MDM2-3AD is resistant to inhibition by mutant p53. (a) The structure of MDM2-3AD. Two extra copies of AD (residues 221 to 280) were inserted. (b) The effect of mutant p53 on MDM2-3AD self-ubiquitination was detected by coexpressing MDM2-3AD and mutant p53 in H1299 cells. MDM2 ubiquitination was analyzed by Ni2+-NTA pulldown and MDM2 Western blotting. (c) H1299 cells were cotransfected with MDM2-3AD, p53, and His6-ubiquitin. MDM2 and p53 ubiquitination was determined by Ni2+-NTA pulldown and Western blotting for MDM2 and p53. (d) H1299 cells were cotransfected with MDM2-3AD and p53-R175H. P53 degradation was determined by Western blotting. (e) MDA-MB-231 (R280K) cells were transiently transfected with the indicated plasmids. The level of endogenous p53 was determined by Western blotting.
- FIG 8
A model of MDM2 inhibition by mutant p53. (a) Wild-type p53 binding to MDM2 is initiated through N-terminal-specific interaction, followed by weak secondary interactions between the AD and RING with the core domain. The AD induces mutant-like conformational change in the core and inhibits p53 DNA binding. The weak AD and RING interactions with the core do not interfere with MDM2 E3 activity and p53 degradation. (b) Mutant p53 interaction with MDM2 is also initiated by specific N-terminal binding. Subsequently, the exposed hydrophobic residues of the mutant p53 core domain bind AD and RING with high avidity, blocking the MDM2 E3 activity. These interactions cooperate with other mechanisms, such as chaperone binding, to inhibit mutant p53 ubiquitination and degradation.
